The present invention relates to methods for treating hypertension, using a factor that stimulates angiogenesis and/or promotes vascular permeability.
Systemic hypertension is the most prevalent cardiovascular disorder in the United States, affecting over 60 million Americans. In spite of increasing public awareness and a rapidly expanding array of antihypertensive medications, hypertension remains one of the leading causes of cardiovascular morbidity and mortality. Hypertension treatments have focused on stimulating the relaxation of the peripheral vasculature (vasodilation), depressing cardiac function, or by stimulating salt transport by blocking epithelial transport of sodium or chloride (diuresis). xe2x80x9cTextbook of Medical Physiologyxe2x80x9d, Guyton and Hall, eds. p. 234 (1996) W. B. Saunders. In addition, adverse metabolic effects have been observed with treatment using certain classes of antihypertensive treatment in coronary disease prevention. xe2x80x9cCecil Textbook of Medicinexe2x80x9d pp. 252-269 (1992) W. B. Saunders. Therefore, there is a need to develop improved methods of treatment of hypertension.
Essential hypertension is the pathological expression of the inability to excrete a dietary sodium load efficiently. The causes for and the mechanism of the development of essentially hypertension are less than clear. According to one of the several possible theories, excreting a sodium load depends on the permeability of the vascular/epithelial barrier in the excreting organs such as the kidney and/or the surface area of the vascular/epithelial structures available for solute flux.
In tissues, the basement membrane serves to separate epithelial cells from blood vessels containing endothelial cells, particularly in the transport or flux of solutes in solution, such as sodium chloride, across basement membranes. Normally, the endothelium is relatively impermeable, limiting the flux of solutes and fluid across the basement membrane against which it is juxtaposed. However, the function of several specialized tissues requires permeable capillary beds to support solute flux. Such functions include the filtration of solutes by the kidney to regulate intravascular volume and maintain normal blood pressure, reabsorption of fluid secretions in the lungs to preserve pulmonary oxygenation, reabsorption of fluids containing solutes in the intestines to provide nutrition, production of cerebrospinal fluid in the choroid plexus of the brain to support and protect the central nervous system, diffusion of nutrients toward non-vascular tissue as occurs in certain portions of the eye or in wound healing, and reabsorption of interstitial fluid from the peritoneum. Impaired transport of solutes across basement membranes contributes to or exacerbates, among other disorders, essential hypertension, kidney disease, acute respiratory disease syndrome, macular ischemia, intestinal inflammatory diseases, meningitis, stroke, ascites, impaired peritoneal dialysis efficiency, and impaired wound healing.
A number of factors may potentially affect solute flux between the endothelial bed and epithelial tissue, including: (1) the metabolic activity of the epithelial cells of a particular tissue; (2) the number of the blood vessels adjacent to the epithelium and its basement membrane; and (3) the porosity or permeability of said blood vessels. Some of the diseases cited above are associated with epithelial cell toxicity, such as acute respiratory distress syndrome and kidney disease or with altered integrity of the capillary blood vessels, as occurs in vasculitis or ischemia. Other disease syndromes, such as essential hypertension have no defined central mechanism. The diseases cited above may be associated with diminished capillary number or altered porosity of the capillary vessel.
Angiogenesis, i.e. the growth of new capillary blood vessels, is a process which is crucial to normal tissue formation and repair. Consequently, factors that are capable of promoting angiogenesis are useful as wound healing agents. Angiogenesis is a multi-step process involving capillary endothelial cell proliferation, migration and tissue penetration. A number of known growth factors, including basic and acidic fibroblast growth factor, transforming growth factor xcex1 and epidermal growth factor, are broadly mitogenic for a variety of cell types as well as being angiogenic and are, therefore, potentially useful in promoting tissue repair. Broad spectrum mitogenicity is desirable in many types of tissue repair applications. There are, however, specific types of tissue repair applications in which it would be desirable to have endothelial cell-specific mitogenic activity, since proliferation of other cell types along with endothelial cells could cause blockage and/or reduced blood flow.
Vascular endothelial growth factor (VEGF) is a secreted endothelial cell mitogen that, when delivered in vivo, promotes new blood vessel formation. The VEGF protein consists of two polypeptide chains, linked by two disulfide bonds. Although the protein is generally described as a homodimer, heterodimeric species have also been reported. Through alternative splicing of the VEGF RNA transcript, five different forms of the monomer chain can be generated, extending 121, 145, 165, 189, and 206 amino acid residues in length. Tischer et al. (1991) J. Biol. Chem. 266:11947-11954; Houck et al. (1991) Mol. Endocrinol. 5:1806-1814; Charnock-Jones et al. (1993) Biol. Reprod. 48:1120-1128; and Neufeld et al. (1996) Cancer Metastasis Rev. 15:153-158. The 121-residue form of VEGF (VEGF121) is unique among the five forms in that it does not bind to heparin-like molecules associated with the extracellular matrix. VEGF121 and the 165-residue form, VEGF165, appear to be the most prevalent forms in vivo.
VEGF is known to stimulate new blood vessel formation by stimulating endothelial cell proliferation and by inducing chemotaxis of endothelial cells. In contrast to other mitogens such as the fibroblast growth factors, VEGF has a much more restricted range of target cell type, and is mitogenic almost exclusively toward endothelial cells. VEGF is also known to enhance vascular permeability and can trigger the relaxation of blood vessels through the release of endothelial nitric oxide. Hariawala et al. (1996) J Surgical Res. 63:77-82; and Sellke et al. (1996) Am. J. Physiol. 271:H713-H720. In addition, VEGF has been shown to regulate the expression of other growth factors and biological mediators and may participate in a growth factor cascade that promotes tissue remodeling and repair.
The activity of VEGF is mediated by interaction with specific membrane receptors on target tissues, most notably the vascular endothelium. Both VEGF121 and VEGF165 are known to interact with two tyrosine kinase receptors: kinase insert domain-containing receptor (KDR; also known as Flk-1), and fms-like tyrosine kinase-1 (Flt-1). deVries et al. (1992) Science 255:989-991; Terman et al. (1992) Biochem. Biophys. Res. Commun. 187:1579-1586; and Millauer et al. (1993) Cell 72:835-846. Both KDR and Flt-1 consist of extracellular ligand-binding domains and intracellular tyrosine kinase domains, the latter being functionally activated upon engagement of VEGF. KDR is found only on endothelial cells, while Flt-1 is found on endothelial cells and monocytes. The angiogenic properties and other known functions of VEGF appear to be mediated via KDR and Flt-1.
Each human kidney comprises about one million nephrons, each capable of forming urine. Each nephron has two major components: a glomerulus, through which large amounts of fluid are filtered from the blood, and a long tubule, in which the filtered fluid is converted into urine. The glomerular capillary has three major layers: the endothelium, a basement membrane, and a layer of epithelial cells. The capillary endothelium is perforated by thousands of small holes called fenestrae.
VEGF can increase the permeability of blood vessels to solutes on a long-term basis by inducing the formation of fenestrations between endothelial cells. Roberts and Palade (1995) J. Cell Sci. 108:2369-2379; and Esser et al. (1998) J. Cell Biol. 140:947-959. In some tissues, such as the renal glomerulus, the glomerular epithelium is known to chronically secrete VEGF, presumably to maintain the fenestrations of the glomerular capillary endothelium. The solute ultrafiltrate that ultimately forms the urine produced by the kidney flows through these fenestrations. The choroid plexus in the brain responsible for producing cerebrospinal fluid and the distal tubule of the kidney, where sodium and potassium are exchanged for the final control of the urine solute concentration also contain fenestrated endothelium adjacent to VEGF-producing epithelium. Proper sodium and potassium exchange in the distal tubule is essential for the maintenance of normal intravascular volume.
Other epithelia known to constitutively produce VEGF include the epithelia of the lung, intestines, and skin. Ferrara and Davis-Smith (1997) Endocrine Rev. 18:4-25; and Monacci et al. (1993) Am. J. Physiol. 264:C995-C1002. Non-epithelial cells that make VEGF are fibroblasts and vascular smooth muscle cells, which secrete VEGF in response to tissue hypoxia, and thus stimulate the formation of new blood vessels. Ferrara and Davis-Smith (1997) Endocrine Rev. 18:4-25.
In contrast to the mesenchymal cells that produce VEGF, hypoxia does not appear to be a stimulus for VEGF production in epithelial cells. Specialized endothelia that express VEGF receptors in the absence of hypoxia include the glomerular and peritubular capillaries of the kidney, the capillaries of the choroid plexus, and endothelia in the intestines, lungs, retina, and heart valve. Little is known about modulation of VEGF secretion by epithelia. In the kidney, it is known that hypoxia is not a signal for VEGF secretion. Krxc3xa4mer et al. (1997) Kidney International 51:444-447.
The present invention provides methods of treating hypertension, particularly essential hypertension. The methods generally involve providing a stimulator of angiogenesis and/or of blood vessel porosity to maintain or correct the transport of solutes, including sodium chloride, and fluid across a basement membrane separating blood vessels or other vessels containing endothelium from epithelial cells. Such transport can be from the blood vessel across the basement membrane to or by the epithelial cells; or it can be from or by epithelia across the basement membrane to blood vessels. Stimulation of vessel number or porosity is used to increase the efficiency or extent of solute transport, thus decreasing blood volume and the concomitant hypertension.
In one aspect, the invention concerns a method for treating essential hypertension, comprising administering to a patient an effective amount of an angiogenic factor, or an agonist thereof. The angiogenic factor can be administered alone or in combination with a further anti-hypertensive agent, such as another angiogenic factor, and preferably is a vascular endothelial growth factor (VEGF) molecule.
The vascular endothelial growth factor is preferably selected from the group consisting of native hVEGF145 (FIG. 7, SEQ ID NO: 2), native hVEGF165 (FIG. 8, SEQ ID NO: 3), native hVEGF189 (FIG. 9, SEQ ID NO: 4), native hVEGF206 (FIG. 10, SEQ ID NO: 5), and agonists of any one of such native VEGF proteins.
In a particularly preferred embodiment, the VEGF molecule lacks the ability to bind heparin, and is, for example, hVEGF121.
The hypertension preferably is salt-dependent hypertension.
In another aspect, the invention concerns an article of manufacture comprising:
a container;
a composition comprising an angiogenic factor or an agonist thereof, in an amount effective in the treatment of hypertension; and
instructions to administer the composition for the treatment of hypertension.
Again, the composition may contain an additional anti-hypertensive agent, e.g. a further angiogenic factor. The angiogenic factor preferably is a VEGF molecule or an agonist thereof.
In a further aspect, the invention concerns a method for identifying an anti-hypertensive agonist of a VEGF molecule comprising testing the ability of a candidate agonist to treat hypertension in a standard animal model of hypertension, in comparison with the VEGF molecule.
The invention further provides compositions for use is the foregoing methods and articles of manufacture. The compositions may contain one or more active ingredients, at least one of which is an angiogenic factor present in an amount effective in the treatment of hypertension. Alternatively, the compositions may comprise one or more polynucleotides comprising nucleotide sequences which encode an angiogenic factor, such as a VEGF, an polypeptide agonist of an angiogenic factor, or a polypeptide factor stimulating the production of an angiogenic factor.